In the following discussion certain articles and methods may be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Molecular arrays typically are precisely-ordered arrangements of large sets of nucleic acid, protein or other molecules immobilized on solid substrates, and are valuable tools in areas of research that require the identification and/or quantification of many molecules in parallel. DNA arrays are the most common type of molecular arrays and have been used in genetic mapping studies, mutational analyses and in genome-wide monitoring of gene expression and have become standard tools in research, diagnostic and clinical applications. Molecular arrays of proteins or peptides are increasingly used in the art and are particularly useful in high throughput screening of molecular interactions such as protein-protein binding and enzymatic activities.
Traditionally, peptide arrays have been made by spotting pre-synthesized peptides on a surface (Salisbury, et al., J. Am. Chem. Soc. 124(50):14868-70 (2002)) or by synthesizing peptides in spots on cellulose filter sheets using standard solid phase peptide synthesis (known as the SPOT method, see Frank, J. Immunol. Methods, 267(1):13-26 (2002)). However, the cost of generating arrays with tens of thousands or more spotted peptides would be astronomically high. Several methods enable direct chemical synthesis of peptides in microarray format, which reduces costs, but these methods still have the major drawback of variability in the quality of the synthesized peptides (Antohe and Cooley, Methods Mol. Biol., 381:299-312 (2007)). Moreover, the direct fabrication process can be very slow and inefficient (Hilpert, et al., Nat. Protoc., 2:1333-49 (2007)).
Recently, methods for peptide array fabrication by in vitro translation of arrayed nucleic acids have been developed, including protein in situ array (PISA) production (He and Taussig, Nucleic Acids Res., 29: e73 (2001)), nucleic acid programmable protein array (NAPPA) production (Ranachandran, et al., Science, 305:86-90 (2004)), DNA to protein array (DAPA) construction (He, Nat. Methods, 5:175-177 (2008)), and arraying of proteins using in situ puromycin capture (Tao and Zhu, Nat. Biotech, 24:1253-1254 (2006)). These approaches utilize individually-synthesized nucleic acid templates rather than individually-synthesized peptides; however, the cost of the nucleic acid templates is often higher than the cost of individual peptides arrayed by traditional methods. Further, diffusion of the peptide products limits the feature density of these types of arrays.
The ability to manufacture large, high-quality, sequence-diverse molecular arrays in a cost effective manner would be of great benefit generally in molecular research, and in the development of diagnostics and therapeutics in particular. The present invention addresses this need.